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Ultrahigh-Throughput Functional Profiling of Microbiota Communities

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Ultrahigh-Throughput Functional Profiling of Microbiota Communities Ultrahigh-throughput functional profiling of microbiota communities Stanislav S. Terekhova,1, Ivan V. Smirnova,b,1, Maja V. Malakhovac, Andrei E. Samoilovc, Alexander I. Manolovc, Anton S. Nazarova, Dmitry V. Danilova, Svetlana A. Dubileyd,e, Ilya A. Ostermane,f, Maria P. Rubtsovae,f, Elena S. Kostryukovac, Rustam H. Ziganshina, Maria A. Kornienkoc, Anna A. Vanyushkinac, Olga N. Bukatoc, Elena N. Ilinac, Valentin V. Vlasovg, Konstantin V. Severinove,h,2, Alexander G. Gabibova,2, and Sidney Altmani,j,2 aShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia; bNational Research University Higher School of Economics, 101000 Moscow, Russia; cFederal Research and Clinical Centre of Physical-Chemical Medicine, Federal Medical and Biological Agency, 119435 Moscow, Russia; dInstitute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia; eSkolkovo Institute of Science and Technology, 143026 Skolkovo, Russia; fDepartment of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia; gInstitute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia; hWaksman Institute for Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08901; iDepartment of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520; and jSchool of Life Sciences, Arizona State University, Tempe, AZ 85287 Contributed by Sidney Altman, July 27, 2018 (sent for review July 9, 2018; reviewed by Satish K. Nair and Meir Wilchek) Microbiome spectra serve as critical clues to elucidate the evolution- screening of antibiotics and prospective probiotic strains. In this ary biology pathways, potential pathologies, and even behavioral work, we adjusted our ultrahigh-throughput (uHT) microfluidic patterns of the host organisms. Furthermore, exotic sources of droplet platform (13) to perform functional screening of wild- microbiota represent an unexplored niche to discover microbial sec- animal microbiota. Our platform allows for packaging the indi- ondary metabolites. However, establishing the bacterial functional- vidual bacterial clones in double emulsion droplets and screening ity is complicated by an intricate web of interactions inside the these microcompartments by FACS. This technique enables functional screening of the cell libraries with an enormous bio- microbiome. Here we apply an ultrahigh-throughput (uHT) microfluidic 8 droplet platform for activity profiling of the entire oral micro- diversity displaying productivity reaching 10 variants per hour. bial community of the Siberian bear to isolate Bacillus strains The critical advantage of the system lies in the possibility of demonstrating antimicrobial activity against Staphylococcus aureus. microbiome functional profiling on a single-cell level. Genome mining allowed us to identify antibiotic amicoumacin A Here we present the analyses of the microbiome collected from East Siberian brown bear (Ursus arctos collaris) obtained (Ami) as responsible for inhibiting the growth of S. aureus.Proteo- immediately after capture in the taiga. We aimed to screen this mics and metabolomics revealed a unique mechanism of Bacillus self- microbiome resource and search for probiotics and physiologi- resistance to Ami, based on a subtle equilibrium of its deactivation cally active compounds. A Bacillus pumilus strain producing an and activation by kinase AmiN and phosphatase AmiO, respectively. We developed uHT quantitative single-cell analysis to estimate anti- biotic efficacy toward different microbiomes and used it to deter- Significance mine the activity spectra of Ami toward human and Siberian bear microbiota. Thus, uHT microfluidic droplet platform activity profiling Analyzing complex microbial communities is the milestone of is a powerful tool for discovering antibiotics and quantifying exter- modern microbiology, calling for “deep functional profiling” nal influences on a microbiome. techniques. While next generation sequencing revolutionized our understanding of microbiota communities, we still lack high- microbiome | microfluidics | deep functional profiling | throughput technologies to precisely determine their functional- antibiotic resistance | single-cell cultivation ity. Here we show how cultivation of individual bacteria inside droplets of microfluidic double water-in-oil-in-water emulsion enables us to isolate the clones with a desired activity. This ap- he latest insights into microbiome revealed close links be- proach allows us not only to select the potent antibiotic producer tween the spectra of coexisting bacteria and progression of T but also to discover a distinct mechanism of self-resistance as well several pathologies in human hosts (1–3). Microbiome spectra as assess its efficiency on entire microbiomes. The outcome of this turned out to be a viable marker of behavior and habits of Homo methodology shows that it could be effectively transferred to sapiens and Neanderthals, as well as the environmental and histor- numerous applications in microbiology and biotechnology. ical conditions they lived in (4). The bactericide properties of microbiome spectra and bacterial coexistence are becoming a hall- Author contributions: S.S.T., I.V.S., S.A.D., E.N.I., V.V.V., K.V.S., A.G.G., and S.A. designed re- BIOCHEMISTRY mark of present-day biomedical investigations in humans (5, 6). search; S.S.T., I.V.S., M.V.M., A.S.N., D.V.D., S.A.D., I.A.O., M.P.R., E.S.K., R.H.Z., M.A.K., A.A.V., Scrutinizing the microbiota of a plethora of organisms is becoming a and O.N.B. performed research; A.E.S., A.I.M., and V.V.V. contributed new reagents/analytic tools; S.S.T., I.V.S., M.V.M., A.S.N., S.A.D., I.A.O., M.P.R., E.S.K., R.H.Z., A.A.V., V.V.V., K.V.S., mainstream of modern microbiology. The microbiota of wild, cap- A.G.G., and S.A. analyzed data; and S.S.T., I.V.S., E.N.I., K.V.S., A.G.G., and S.A. wrote the paper. tive, and domesticated animals as well as birds, reptiles, and gene- – – Reviewers: S.K.N., University of Illinois at Urbana Champaign; and M.W., Weizmann In- modified animal models (6 10) were analyzed, offering valuable stitute of Science. answers to the long-standing issues of biology. Microbiomes have The authors declare no conflict of interest. also come into the limelight of evolutionary studies (11). This open access article is distributed under Creative Commons Attribution-NonCommercial- The revolution in screening technologies complemented by NoDerivatives License 4.0 (CC BY-NC-ND). that of functional and structural analyses of large arrays of Data deposition: The data reported in this paper have been deposited in the GenBank microbiota species on a single-cell level (12) allows us to isolate database, https://www.ncbi.nlm.nih.gov/genbank/ (accession nos. QENN00000000 and and characterize clones with different activities, such as micro- QENO00000000). bial killers, antifungi and antiparasite drugs, as well as probiotic 1S.S.T. and I.V.S. contributed equally to this work. bacterial strains. Microbiota of wild animals is an under- 2To whom correspondence may be addressed. Email: [email protected], estimated resource for this type of screenings. The ability of wild [email protected], or [email protected]. animals to thrive while surrounded by aggressive microorganisms This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. may be partially mediated by their microbiota, making this kind 1073/pnas.1811250115/-/DCSupplemental. of microbiota a potentially attractive niche for a targeted Published online September 4, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1811250115 PNAS | September 18, 2018 | vol. 115 | no. 38 | 9551–9556 Downloaded by guest on September 29, 2021 ABMicrobiota Waste Exotic source Target Killer Cocultivation Coencapsulation and staining Sorting Fig. 1. (A) Principal scheme of bacteria isolation demonstrating antimicrobial activity against the target S. aureus from oral microbiota of the Siberian bear using the uHT microfluidic droplet platform. The isolated microbiota was coencapsulated with the target pathogen producing a GFP reporter. After in droplet cocultivation, the bacteria were stained and sorted for isolating the droplets with a high initial target load, low GFP level, and high metabolic activity. (B) MDE droplets with coencapsulated target S. aureus cells and microbiota species after in droplet cocultivation. (Scale bar: 50 μm.) unstable antibiotic amicoumacin A (Ami) was isolated, enabling for bacteria inhibiting the growth of pathogenic Staphylococcus us to identify the B. pumilus Ami biosynthetic gene cluster and aureus cells using a microfluidic platform (Fig. 1). It is based on discover a crucial role of Ami kinase/phosphatase in the regu- cocultivation of oral microbiota members with the target S. aureus lation of Ami production. We than applied uHT screening to strain producing GFP reporter in droplets of microfluidic double quantify the external influence on the microbiome diversity, thus water-in-oil-in-water emulsion (MDE). obtaining a detailed description of Ami activity spectra toward The combination of three independent fluorescent signals was human and Siberian bear microbiota. used to isolate MDE droplets by FACS. The isolated droplets had to conform to the following criteria simultaneously; they exhibited Results ahighinitialS. aureus load and a low S. aureus count after in Microfluidic Selection of Antibiotic Activity. The saliva samples
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